The present invention relates to novel enzyme variants useful in a variety of cleaning compositions, and the genes encoding such enzyme variants.
Enzymes make up the largest class of naturally occurring proteins. Each class of enzyme generally catalyzes (accelerates a reaction without being consumed) a different kind of chemical reaction. One class of enzymes known as proteases, are known for their ability to hydrolyze (break down a compound into two or more simpler compounds with the uptake of the H and OH parts of a water molecule on either side of the chemical bond cleaved) other proteins. This ability to hydrolyze proteins has been taken advantage of by incorporating naturally occurring and protein engineered proteases as an additive to laundry detergent preparations. Many stains on clothes are proteinaceous and wide-specificity proteases can substantially improve removal of such stains.
Unfortunately, the efficacy level of these proteins in their natural, bacterial environment, frequently does not translate into the relatively unnatural wash environment. Specifically, protease characteristics such as thermal stability, pH stability, oxidative stability and substrate specificity are not necessarily optimized for utilization outside the natural environment of the enzyme.
The amino acid sequence of the protease determines the characteristics of the protease. A change of the amino acid sequence of the protease may alter the properties of the enzyme to varying degrees, or may even inactivate the enzyme, depending upon the location, nature and/or magnitude of the change in the amino acid sequence. Several approaches have been taken to alter the wild-type amino acid sequence of proteases in an attempt to improve their properties, with the goal of increasing the efficacy of the protease in the wash environment. These approaches include altering the amino acid sequence to enhance thermal stability and to improve oxidation stability under quite diverse conditions.
Despite the variety of approaches described in the art, there is a continuing need for new effective variants of proteases useful for cleaning a variety of surfaces.
It is an object of the present invention to provide subtilisin Carlsberg enzyme variants having improved hydrolysis versus the wild-type of the enzyme.
It is also an object of the present invention to provide cleaning compositions comprising these subtilisin enzyme variants.
The present invention relates to subtilisin Carlsberg variants having a modified amino acid sequence of wild-type subtilisin Carlsberg amino acid sequence, the wild-type amino acid sequence comprising a first loop region, a second loop region, a third loop region, a fourth loop region and a fifth loop region; wherein the modified amino acid sequence comprises different amino acids than that occurring in wild-type subtilisin Carlsberg (i.e., substitution) at specifically identified positions in one or more of the loop regions whereby the subtilisin Carlsberg variant has decreased adsorption to, and increased hydrolysis of, an insoluble substrate as compared to the wild-type subtilisin Carlsberg. The present invention also relates to the genes encoding such subtilisin Carlsberg variants. The present invention also relates to compositions comprising such subtilisin Carlsberg variants for cleaning a variety of surfaces.
I. Subtilisin Variants
This invention pertains to subtilisin enzymes, in particular subtilisin Carlsberg, that have been modified by mutating the various nucleotide sequences that code for the enzyme, thereby modifying the amino acid sequence of the enzyme. The modified subtilisin enzymes (hereinafter, xe2x80x9csubtilisin Carlsberg variantsxe2x80x9d) of the present invention have decreased adsorption to and increased hydrolysis of an insoluble substrate as compared to the wild-type subtilisin. The present invention also pertains to the mutant genes encoding for such subtilisin Carlsberg variants.
The subtilisin enzymes of this invention belong to a class of enzymes known as proteases. A protease is a catalyst for the cleavage of peptide bonds. One type of protease is a serine protease. A serine protease is distinguished by the fact that there is an essential serine residue at the active site.
The observation that an enzyme""s rate of hydrolysis of soluble substrates increases with enzyme concentration is well documented. It would therefore seem plausible that for surface bound substrates, such as is encountered in many cleaning applications, the rate of hydrolysis would increase with increasing surface concentration. This has been shown to be the case. (Brode, P.F. III and D. S. Rauch, LANGMUIR, xe2x80x9cSubtilisin BPN"": Activity on an Immobilized Substratexe2x80x9d, Vol. 8, pp. 1325-1329 (1992)). In fact, a linear dependence of rate upon surface concentration was found for insoluble substrates when the surface concentration of the enzyme was varied. (Rubingh, D. N. and M. D. Bauer, xe2x80x9cCatalysis of Hydrolysis by Proteases at the Protein-Solution Interface,xe2x80x9d in POLYMER SOLUTIONS, BLENDS AND INTERFACES, Ed. by I. Noda and D. N. Rubingh, Elsevier, p. 464 (1992)). Surprisingly, when seeking to apply this principle in the search for variant proteases which give better cleaning performance, we did not find that enzymes which adsorb more give better performance. In fact, we surprisingly determined the opposite to be the case: decreased adsorption by an enzyme to a substrate resulted in increased hydrolysis of the substrate (i.e., better cleaning performance).
While not wishing to be bound by theory, it is believed that improved performance, when comparing one variant to another, is a result of the fact that enzymes which adsorb less are also less tightly bound and therefore more highly mobile on the surface from which the insoluble protein substrate is to be removed. At comparable enzyme solution concentrations, this increased mobility is sufficient to outweigh any advantage that is conferred by delivering a higher concentration of enzyme to the surface.
The mutations described herein are designed to change (i.e., decrease) the adsorption of the enzyme to surface-bound soils. In subtilisin Carlsberg, certain amino acids form exterior loops on the enzyme molecule. For purposes of discussion, these loops shall be referred to as first, second, third, fourth and fifth loop regions. Specifically, positions 5845 form the first loop region; positions 94-106 form the second loop region; positions 125-132 form the third loop region; positions 153-166 form the fourth loop region; positions 186-190 form the fifth loop region; and positioins 199-219 form the sixth loop region (position numbering analagous to positions in the amino acid sequence for wild-type subtilisin subtilisin Carlsberg (SEQ ID NO:1)).
It believed that these loop regions play a significant role in the adsorption of the enzyme molecule to a surface-bound peptide, and specific mutations in one or more of these loop regions will have a significant effect on this adsorption. While not wishing to be bound by theory, it is believed that the loop regions are important to the adsorption of the subtilisin Carlsberg molecule for at least two reasons. First, the amino acids which comprise the loop regions can make close contacts with any surfaces to which the molecule is exposed. Second, the proximity of the loop regions to the active-site and binding pocket of the subtilisin Carlsberg molecule gives them a role in the catalytically productive adsorption of the enzyme to surface-bound substrates (peptides/protein soils).
As used herein, xe2x80x9cvariantxe2x80x9d means an enzyme having an amino acid sequence which differs from that of wild-type.
As used herein, xe2x80x9cmutant subtilisin Carlsberg genexe2x80x9d means a gene coding for a subtilisin Carlsberg variant.
As used herein, xe2x80x9cwild-type subtilisin Carlsbergxe2x80x9d refers to a subtilisin enzyme represented by SEQ ID NO:1. The amino acid sequence for subtilisin Carlsberg is further described by Smith, E. L., Delange, R. J Evans, W. H., is Landon, M., and Markland, F. S., J. BIOL. CHEM., Vol. 243, pp. 2184-2191 (1968), incorporated herein by reference.
As used herein, the term xe2x80x9csubtilisin Carlsberg wild-type amino acid sequencexe2x80x9d encompasses SEQ ID NO:1 as well as SEQ ID NO:1 having modifications to the amino acid sequence other than at any of positions 58-65, 94-106, 125-132, 153-166, 186-190 and 199-219.
As used herein, xe2x80x9cmore hydrophilic amino acidxe2x80x9d refers to any other amino acid having greater hydrophilicity than a subject amino acid with reference to the hydrophilicity table below. The following hydrophilicity table (Table 1) lists amino acids in descending order of increasing hydrophilicity (see Hopp, T. P., and Woods, K. R., xe2x80x9cPrediction of Protein Antigenic Determinants from Amino Acid Sequencesxe2x80x9d, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCE USA, Vol. 78, pp. 3824-3828, 1981, incorporated herein by reference).
Table 1 also indicates which amino acids carry a charge (this characteristic being based on a pH of from about 8-9). The positively charged amino acids are Arg and Lys, the negatively charged amino acids are Glu and Asp, and the remaining amino acids are neutral. In a preferred embodiment of the present invention, the substituting amino acid is either neutral or negatively charged, more preferably negatively charged (i.e., Glu or Asp).
Therefore, for example, the statement xe2x80x9csubstitute Gln with an equally or more hydrophilic amino acid which is neutral or has a negative chargexe2x80x9d means Gin would be substituted with Asn (which is equally hydrophilic to Gln), or Ser, Glu or Asp (which are more hydrophilic than Gin); each of which are neutral or have a negative charge, and have a greater hydrophilicity value as compared to Gin. Likewise, the statement xe2x80x9csubstitute Pro with a more hydrophilic amino acid which is neutral or has a negative chargexe2x80x9d means Pro would be substituted with Gln, Asn, Ser, Glu or Asp.
In one embodiment of the present invention, the subtilisin Carlsberg variant has a modified amino acid sequence of subtilisin Carlsberg wild-type amino acid sequence, wherein the wild-type amino acid sequence comprises a substitution at one or more positions in one or more of the first loop region, the second loop region, the third loop region, the fourth loop region, the fifth loop region or the sixth loop region; whereby the subtilisin Carlsberg variant has decreased adsorption to, and increased hydrolysis of, an insoluble substrate as compared to the wild-type subtilisin Carlsberg.
In a preferred embodiment of the present invention, the substituting amino acid for one or more of the positions in one or more of the loop regions is, with reference to Table 1, neutral or negatively charged and equally or more hydrophilic, preferably more hydrophilic, than the amino acid at the subject position in the wild-type amino acid sequence.
A. Substitutions in the First Loop Region
When a substitution occurs in the first loop region, the substitution occurs at one or more of positions 58, 59, 60, 61, 62, 64 or 65.
When a substitution occurs at position 58, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, Pro or Ser.
When a substitution occurs at position 59, the substituting amino acid is Glu.
When a substitution occurs at position 60, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser.
When a substitution occurs at position 61, the substituting amino acid is Asp, Gln, Glu or Ser.
When a substitution occurs at position 62, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser.
When a substitution occurs at position 64, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser.
When a substitution occurs at position 65, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, Pro or Ser.
B. Substitutions in the Second Loop Region
When a substitution occurs in the second loop region, the substitution occurs at one or more of positions 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105 or 106.
When a substitution occurs at position 94, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Met, Pro, Ser or Thr.
When a substitution occurs at position 95, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Met, Pro, Ser, Thr or Val.
When a substitution occurs at position 96, the substituting amino acid is Asp, Gln, Glu, Ser.
When a substitution occurs at position 97, the substituting amino acid is Asp or Glu.
When a substitution occurs at position 98, the substituting amino acid is Asp or Glu.
When a substitution occurs at position 99, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser.
When a substitution occurs at position 100, the substituting amino acid is Asp or Glu.
When a substitution occurs at position 101, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser.
When a substitution occurs at position 102, the substituting amino acid is Asp or Glu.
When a substitution occurs at position 103, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Pro, Ser, Thr or Val.
When a substitution occurs at position 104, the substituting amino acid is Asp or Glu.
When a substitution occurs at position 105, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser.
When a substitution occurs at position 106, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Leu, Met, Pro, Ser, Thr or Val.
C. Substitutions in the Third Loop Region
When a substitution occurs in the third loop region, the substitution occurs at one or more of positions 125,126,127, 128, 129, 130, 131 or 132.
When a substitution occurs at position 125, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Met, Pro, Ser, Thr or Val.
When a substitution occurs at position 126, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser.
When a substitution occurs at position 127, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser.
When a substitution occurs at position 128, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, His, Pro, Ser or Thr.
When a substitution occurs at position 129, the substituting amino acid is Asp or Glu.
When a substitution occurs at position 130, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser.
When a substitution occurs at position 131, the substituting amino acid is Asp or Glu.
When a substitution occurs at position 132, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, Pro or Ser.
D. Substitutions in the Fourth Loop Region
When a substitution occurs in the fourth loop region, the substitution occurs at one or more of positions 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165 or 166.
When a substitution occurs at position 153, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser.
When a substitution occurs at position 154, the substituting amino acid is Asp, Gln, Glu or Ser.
When a substitution occurs at position 155, the substituting amino acid is Asp or Glu.
When a substitution occurs at position 156, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser.
When a substitution occurs at position 157, the substituting amino acid is Asp, Gln, Glu, Ser.
When a substitution occurs at position 158, the substituting amino acid is Asp or Glu.
When a substitution occurs at position 159, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser.
When a substitution occurs at position 160, the substituting amino acid is Asp or Glu.
When a substitution occurs at position 161, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, Pro or Ser.
When a substitution occurs at position 162, the substituting amino acid is Asp, Gln, Glu or Ser.
When a substitution occurs at position 163, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, Pro or Ser.
When a substitution occurs at position 164, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Met, Pro, Ser, Thr or Val.
When a substitution occurs at position 165, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser. and
When a substitution occurs at position 166, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Pro, Ser, Thr or Val.
E. Substitutions in the Fifth Loop Region
When a substitution occurs in the fifth loop region, the substitution occurs at one or more of positions 186, 187, 188, 189 or 190.
When a substitution occurs at position 186, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, His, Pro, Ser and Thr.
When a substitution occurs at position 187, the substituting amino acid is Asp or Glu.
When a substitution occurs at position 188, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Pro, Ser, Thr, Tyr or Val.
When a substitution occurs at position 189, the substituting amino acid is Asp or Glu.
When a substitution occurs at position 190, the substituting amino acid is Asp or Glu.
F. Substitutions in the Sixth Loop Region
When a substitution occurs in the sixth loop region, the substitution occurs at one or more of positions 199, 200, 201, 202, 203, 204, 205, 206, 207,208, 209, 210,211, 212,213,214,215, 216,217,218 or 219.
When a substitution occurs at position 199, the substituting amino acid is Asn Asp, gin, Glu, Gly, His, Pro, Ser or Thr.
When a substitution occurs at position 200, the substituting amino acid is Asn, Asp, Gln, Glu, Gly or Ser.
When a substitution occurs at position 201, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser.
When a substitution occurs at position 202, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, His, Pro, Ser or Thr.
When a substitution occurs at position 203, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser.
When a substitution occurs at position 204, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Met, Pro, Ser or Thr.
When a substitution occurs at position 205, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Pro, Ser, Thr or Val.
When a substitution occurs at position 206, the substituting amino acid is Asp or Glu.
When a substitution occurs at position 207, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, Pro or Ser.
When a substitution occurs at position 208, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Pro, Ser, Thr or Val.
When a substitution occurs at position 209, the substituting amino acid is Asn, Asp, Gln, Glu, Gly or Ser.
When a substitution occurs at position 210, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, Pro or Ser.
When a substitution occurs at position 211, the substituting amino acid is Asp, Gln, Glu or Ser.
When a substitution occurs at position 212, the substituting amino acid is Asn, Asp, Gln, Glu, Gy, Pro or Ser.
When a substitution occurs at position 213, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Met, Pro, Ser, Thr or Val.
When a substitution occurs at position 214, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, His, Pro, Ser or Thr.
When a substitution occurs at position 215, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, Pro or Ser.
When a substitution occurs at position 216, the substituting amino acid is Ala, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Met, Pro, Ser, Thr or Val.
When a substitution occurs at position 217, the substituting amino acid is Asp, Gln, Glu or Ser.
When a substitution occurs at position 218, the substituting amino acid is Asn, Asp, Gln, Glu, Pro or Ser.
When a substitution occurs at position 219, the substituting amino acid is Asn, Asp, Gln, Glu, Gly, Pro or Ser.
G. Preparation of enzyme variants